Streptococcus pneumoniae 37-KDA surface adhesin a protein and nucleic acids coding therefor

ABSTRACT

The invention provides a nucleic acid encoding the 37-kDa protein from Streptococcus pneumoniae. Also provided are isolated nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. The invention also provides purified polypeptides encoded by the nucleic acid encoding the 37-kDa protein from and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Also provided are antibodies which selectively binds the polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Also provided are vaccines comprising immunogenic polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Further provided is a method of detecting the presence of Streptococcus pneumoniae in a sample comprising the steps of contacting a sample suspected of containing Streptococcus pneumoniae with nucleic acid primers capable of hybridizing to a nucleic acid comprising a portion of the nucleic acid encoding the 37-kDa protein, amplifying the nucleic acid and detecting the presence of an amplification product, the presence of the amplification product indicating the presence of Streptococcus pneumoniae in the sample. Further provided are methods of detecting the presence of Streptococcus pneumoniae in a sample using antibodies or antigens, methods of preventing and treating Streptococcus pneumoniae infection in a subject.

This application is a continuation-in-part of Ser. No. 08/222,179, filedApr. 4, 1994 now abandoned, which is a continuation-in-part of Ser. No.07/791,377, filed Sep. 17, 1991, now U.S. Pat. No. 5,422,427, both ofwhich are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the 37-kDa Streptococcus pneumoniae surfaceadhesin A protein. Specifically, the invention relates to an isolatednucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae, tounique fragments of the nucleic acid encoding the 37-kDa protein ofStreptococcus pneumoniae, and to the polypeptides encoded by thosenucleic acids. The invention further relates to antibodies to thosepolypeptides, and to methods of detecting the presence of Streptococcuspneumoniae, methods of preventing Streptococcus pneumoniae infection,and methods of treating a Streptococcus pneumoniae infection.

2. Background Art

Pneumococcal disease continues to be a leading cause of sickness anddeath in the United States and throughout the world. Both the lack ofefficacy of the currently used polysaccharide vaccines in children under2 years of age and their variable serotype-specific efficacy amongvaccinated individuals, have prompted manufacturers to investigatealternative vaccine formulations that do not require the use of multiplecapsular polysaccharides. One current approach under consideration isthe use of immunogenic species-common proteins as vaccine candidates.These proteins could be used in combination with other immunogenicproteins or as protein carriers in a protein-polysaccharide oroligosaccharide conjugate vaccine. An effective vaccine that included acommon protein could eliminate the need for formulations based onmultiple capsular polysaccharides (as in the 23-valent polysaccharidevaccine) by offering a broader range of protection against a greaternumber of serotypes. Additionally, a protein-based vaccine would beT-cell dependent and provide a memory response, resulting in a moreefficacious vaccine.

Of the reported pneumococcal proteins, only pneumolysin and thepneumococcal surface protein A (PspA) have been extensively examined fortheir suitability as vaccine candidates. While both have been shown tobe partially protective in mice (Paton et al. 1983. "Effect ofimmunization with pneumolysin on survival time of mice challenged withStreptococcus pneumoniae." Infect. Immun. 40:548-552 and McDaniel et al.1991. "PspA, a surface protein of Streptococcus pneumoniae, is capableof eliciting protection against pneumococci of more than one capsulartype." Infect. Immun. 59:222-228), there are disadvantages to their useas vaccine immunogens. Pneumolysin, although well conserved amongpneumococci, has been shown to have strong toxic effects in its nativestate (AlonsoDeVelasco et al. 1995. "Streptococcus pneumoniae: Virulencefactors, pathogenesis, and vaccines." Microbiol. Rev. 59:591-603).Recombinant derivatives of reduced toxicity have been produced, andwhile they show promise in animal protection studies (Alexander et al.1994. "Immunization of mice with pneumolysin toxoid confers asignificant degree of protection against at least nine serotypes ofStreptococcus pneumoniae. Infect. Immun. 62:5683-5688) the problem ofmaintaining maximal immunogenicity and eliminating toxicity to humans isstill in question. PspA, on the other hand, is serologically andstructurally heterogeneous. (Crain et al. 1990. "Pneumococcal surfaceprotein A (PspA) is serologically highly variable and is expressed byall clinically important capsular serotypes of Streptococcuspneumoniae." Infect. Immun. 58:3293-3299). Its use in vaccineformulations would require multiple PspA types, thus increasing thecomplexity of vaccine preparation.

An immunogenic species-common protein has been identified fromStreptococcus pneumoniae. (Russell et al. 1990. "Monoclonal antibodyrecognizing a species-specific protein from Streptococcus pneumoniae."J. Clin. Microbiol. 28:2191-2195 and U.S. Pat. No. 5,422,427 in whichthe 37-kDa protein is referred to as pneumococcal fimbrial protein A).The 37-kDa S. pneumoniae protein has been the focus of several studiesand has been designated pneumococcal surface adhesin protein A (PsaA).Immunoblot analysis studies using anti-PsaA monoclonal antibody showedthat PsaA is common to all 23 pneumococcal vaccine serotypes (Russell etal. 1990). Enzyme-linked-immunosorbent assay studies have indicated thatpatients with pneumococcal disease show an antibody increase inconvalescent-phase serum to PsaA compared with acute-phase serumantibody levels (Tharpe et al. 1995. "Purification and seroreactivity ofpneumococcal surface adhesin A (PsaA)." Clin. Diagn. Lab. Immunol.3:227-229 and Tharpe et al. 1994. "The utility of a recombinant proteinin an enzyme immunoassay for antibodies against Streptococcuspneumoniae." abstr. V-2, p. 617. 1994. American Society forMicrobiology, Washington, D.C.). Additionally, a limited in vivoprotection study showed that antibodies to the 37-kDa protein protectmice from lethal challenge (Talkington et al. 1996. "Protection of miceagainst fatal pneumococcal challenge by immunization with pneumococcalsurface adhesin A (PsaA)." Microbial Pathogenesis 21:17-22).

The gene encoding PsaA from S. pneumoniae strain R36A (an unencapsulatedstrain) has been cloned in Escherichia coli and sequenced, but thisserotype does not contain a 37 kDa protein encoding nucleic acid that ishighly conserved among the various serotypes. (Sampson et al. 1994."Cloning and nucleotide sequence analysis of psaA, the Streptococcuspneumoniae gene encoding a 37-kilodalton protein homologous topreviously reported Streptococcus sp. adhesins." Infect. Immun.62:319-324). This particular nucleic acid and polypeptide, therefore,are of limited value for use as diagnostic reagents, in infectionprevention, in infection treatment, or in vaccine development.

Sequence conservation is a necessary requirement for a candidatespecies-common vaccine. At present, there are no studies that haveinvestigated the sequence conservation of the psaA gene amongpneumococcal types, specifically among encapsulated pneumococci whichcause the vast majority of serious disease. Therefore, a need exists toinvestigate the conservation of the gene in order to provide apolypeptide which can serve as a vaccine for multiple strains ofStreptococcus pneumoniae. The present invention fulfills that need byanalyzing psaA genes from the 23 serotypes in the 23-valentpolysaccharide vaccine and by providing a polypeptide and antibodies tothat polypeptide which are conserved among the S. pnuemoniae serotypesand which confer protection to Streptococcus pneumoniae infection.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to anisolated nucleic acid encoding the 37-kDa protein of Streptococcuspneumoniae as set forth in the Sequence Listing as SEQ ID NO:2. Theinvention also provides unique fragments of at least 10 nucleotides ofthe nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, whichcan be used in methods to detect the presence of Streptococcuspneumoniae in a sample and as immunogenic vaccines.

The invention further provides a purified polypeptide encoded by thenucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae asset forth in the Sequence Listing as SEQ ID NO:1, which can be used asimmunogenic vaccines.

In another aspect, the invention provides purified antibodies which bindto the 37-kDa protein of Streptococcus pneumoniae or fragments thereof.These antibodies can be used in methods to detect the presence ofStreptococcus pneumoniae in a sample and in therapeutic and prophylacticmethods.

The advantages of the invention will be realized and attained by meansof the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this application pertains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included therein.

Before the present compounds and methods are disclosed and described, itis to be understood that this invention is not limited to specificproteins, specific methods, or specific nucleic acids, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms "a," "an," and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "a nucleic acid" includes multiple copies of the nucleicacid and can also include more than one particular species of nucleicacid molecule.

Nucleic Acids

In one aspect, the invention provides an isolated nucleic acid encodingthe 37-kDa protein of Streptococcus pneumoniae as set forth in theSequence Listing as SEQ ID NO:2. The term "isolated" refers to a nucleicacid which is essentially separated from other genes that naturallyoccur in S. pneumoniae. In one embodiment, the present inventionprovides an isolated nucleic acid encoding the 37-kDa protein ofStreptococcus pneumoniae wherein the nucleic acid is the nucleic acidset forth in the Sequence Listing as SEQ ID NO:1.

The nucleic acids of the present invention can include the positiveand/or negative RNA strand as well as the sense and/or nonsense DNAstrand, or any combinations thereof. These nucleic acids include thegenomic DNA fragment encoding the 37-kDa protein and any subgenomicnucleic acids, including DNA and RNA, in the organism encoding all, or afragment of the 37-kDa protein. The nucleic acid can also be modified,such as nucleic acids containing methylated bases.

This nucleic acid can comprise the coding sequence for the 37-kDaprotein itself, or the coding sequence with the gene's upstream anddownstream regulatory sequences, or any combination thereof This nucleicacid can, for example, comprise a DNA and include its own promoter, oranother promoter can be operatively linked to the nucleic acid such thatthe coding sequence of the 37-kDa protein is expressed. Alternatively,the nucleic acid can comprise an RNA such that the RNA is translated andthe resulting polypeptide comprises the 37-kDa protein. Thereforesequences normally found associated with the 37-kDa protein codingsequence, such as the promoter, and the translation signals, can besubstituted, deleted, or modified.

An isolated nucleic acid comprising a unique fragment of at least 10nucleotides of the nucleic acid set forth in the Sequence Listing as SEQID NO:1 is also provided. Unique fragments, as used herein means anucleic acid of at least 10 nucleotides that is not identical to anyother known nucleic acid sequence. Examples of the sequences of at least10 nucleotides that are unique to the nucleic acid set forth in theSequence Listing as SEQ ID NO:1 can be readily ascertained by comparingthe sequence of the nucleic acid in question to sequences catalogued inGenBank, or any other sequence database, using computer programs such asDNASIS (Hitachi Engineering, Inc.) or Word Search or FASTA of theGenetics Computer Group (GCG) (Madison, Wis.), which search thecatalogued nucleotide sequences for similarities to the nucleic acid inquestion. If the sequence does not match any of the known sequences, itis unique. For example, the sequence of nucleotides 1-10 can be used tosearch the databases for an identical match. If no matches are found,then nucleotides 1-10 represent a unique fragment. Next, the sequence ofnucleotides 2-11 can be used to search the databases, then the sequenceof nucleotides 3-13, and so on up to nucleotides 1320 to 1330 of thesequence set forth in the Sequence Listing as SEQ ID NO:1. The same typeof search can be performed for sequences of 11 nucleotides, 12nucleotides, 13 nucleotides, etc. The possible fragments range from 10nucleotides in length to 1 nucleotide less than the sequence set forthin the Sequence Listing as SEQ ID NO:1. These unique nucleic acids, aswell as degenerate nucleic acids can be used, for example, as primersfor amplifying nucleic acids from other strains of Streptococcuspneumoniae in order to isolate allelic variants of the 37-kDa protein,or as primers for reverse transcription of 37-kDa protein RNA, or asprobes for use in detection techniques such as nucleic acidhybridization. One skilled in the art will appreciate that even though anucleic acid of at least 10 nucleotides is unique to a specific gene,that nucleic acid fragment can still hybridize to many other nucleicacids and therefore be used in techniques such as amplification andnucleic acid detection.

Also provided are nucleic acids which encode allelic variants of the37-kDa protein of S. pneumoniae set forth in the Sequence Listing as SEQID NO:2, and those proteins. As used herein, the term "allelicvariations" or "allelic variants" is used to describe the same, orsimilar 37-kDa pneumococcal surface adhesin proteins that are divergedfrom the 37-kDa Streptococcus pneumoniae protein set forth in theSequence Listing as SEQ ID NO:2 by less than 15% in their correspondingamino acid identity. In another embodiment, these allelic variants areless than 10% divergent in their corresponding amino acid identity. Inanother embodiment, these allelic variants are less than 7% divergent intheir corresponding amino acid identity. In another embodiment, theseallelic variants are less than 5% divergent in their corresponding aminoacid identity. In another embodiment, these allelic variants are lessthan 3% divergent in their corresponding amino acid identity. In anotherembodiment, these allelic variants are less than 2% divergent in theircorresponding amino acid identity. In yet another embodiment, theseallelic variants are less than 1% divergent in their corresponding aminoacid identity. These amino acids can be substitutions within the aminoacid sequence set forth in the Sequence Listing as SEQ ID NO:2, they canbe deletions from the amino acid sequence set forth in the SequenceListing as SEQ ID NO:2, and they can be additions to the amino acidsequence set forth in the Sequence Listing as SEQ ID NO:2.

The homology between the protein coding region of the nucleic acidencoding the allelic variant of the 37-kDa protein is preferably lessthan 20% divergent from the region of the nucleic acid set forth in theSequence Listing as SEQ ID NO:1 encoding the 37-kDa protein. In anotherembodiment, the corresponding nucleic acids are less than 15% divergentin their sequence identity. In another embodiment, the correspondingnucleic acids are less than 10% divergent in their sequence identity. Inanother embodiment, the corresponding nucleic acids are less than 7%divergent in their sequence identity. In another embodiment, thecorresponding nucleic acids are less than 5% divergent in their sequenceidentity. In another embodiment, the corresponding nucleic acids areless than 4% divergent in their sequence identity. In anotherembodiment, the corresponding nucleic acids are less than 3% divergentin their sequence identity. In another embodiment, the correspondingnucleic acids are less than 2% divergent in their sequence identity. Inyet another embodiment, the corresponding nucleic acids are less than 1%divergent in their sequence identity. In particular, the nucleic acidvariations can create up to about 15% amino acid sequence variation fromthe protein set forth in the Sequence Listing as SEQ ID NO:2.

One skilled in the art will appreciate that nucleic acids encodinghomologs or allelic variants of the 37-kDa protein set forth in theSequence Listing as SEQ ID NO:2 can be isolated from relatedgram-positive bacteria in a manner similar to that used to isolate thenucleic acid set forth in the Sequence Listing of the present inventionas SEQ ID NO:1. For example, given the sequence of the primers used toamplify the nucleic acid set forth in the sequence listing as SEQ IDNO:1, one can use these or similar primers to amplify a homologous genefrom related gram-positive bacteria.

Alternatively, allelic variants can be identified and isolated bynucleic acid hybridization techniques. Probes selective to the nucleicacid set forth in the Sequence Listing as SEQ ID NO:1 can be synthesizedand used to probe nucleic acid from the various serotypes of S.pneumoniae. High sequence complementarity and stringent hybridizationconditions can be selected such that the probe selectively hybridizes toallelic variants of the sequence set forth in the Sequence Listing asSEQ ID NO:1. For example, the selectively hybridizing nucleic acids ofthe invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and99% complementarity with the segment of the sequence to which ithybridizes. The nucleic acids can be at least 10, 12, 50, 100, 150, 200,300, 500, 750, or 1000 nucleotides in length. Thus, the nucleic acid canbe a coding sequence for the 37-kDa protein or fragments thereof thatcan be used as a probe or primer for detecting the presence of S.pneumoniae. If used as primers, the invention provides compositionsincluding at least two nucleic acids which hybridize with differentregions so as to amplify a desired region. Depending on the length ofthe probe or primer, target region can range between 70% complementarybases and full complementarity and still hybridize under stringentconditions. For example, for the purpose of diagnosing the presence ofan allelic variant of the sequence set forth in the Sequence Listing asSEQ ID NO:1, the degree of complementarity between the hybridizingnucleic acid (probe or primer) and the sequence to which it hybridizesis at least enough to distinguish hybridization with a nucleic acid fromunrelated bacteria. The invention provides examples of nucleic acidsunique to SEQ ID NO:1 in the Sequence Listing so that the degree ofcomplementarity required to distinguish selectively hybridizing fromnonselectively hybridizing nucleic acids under stringent conditions canbe clearly determined for each nucleic acid. One skilled in the art willappreciate that sequences can be added to either one end or both ends ofunique fragments, for example, to aid subsequent cloning, expression, ordetection of the fragment.

"Stringent conditions" refers to the washing conditions used in ahybridization protocol. In general, the washing conditions should be acombination of temperature and salt concentration chosen so that thedenaturation temperature is approximately 5°-20° C. below the calculatedT_(m) of the nucleic acid hybrid under study. The temperature and saltconditions are readily determined empirically in preliminary experimentsin which samples of reference DNA immobilized on filters are hybridizedto the probe or protein coding nucleic acid of interest and then washedunder conditions of different stringencies. The T_(m) of such anoligonucleotide can be estimated by allowing 2° C. for each A or Tnucleotide, and 4° C. for each G or C. For example, an 18 nucleotideprobe of 50% G+C would, therefore, have an approximate T_(m) of 54° C.

In another aspect, the present invention provides an isolated nucleicacid comprising the nucleic acid as set forth in the Sequence Listing asSEQ ID NO:3.

In another aspect, the present invention provides an isolated nucleicacid comprising the nucleic acid as set forth in the Sequence Listing asSEQ ID NO:4.

The nucleic acid encoding a 37-kDa protein may be obtained by any numberof techniques known to one skilled in the art. One method is tosynthesize a recombinant nucleic acid molecule. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein or regulatory regionare readily obtainable through automated DNA synthesis. A nucleic acidfor one strand of a double-stranded molecule can be synthesized andhybridized to its complementary strand. One can design theseoligonucleotides such that the resulting double-stranded molecule haseither internal restriction sites or appropriate 5' or 3' overhangs atthe termini for cloning into an appropriate vector. Double-strandedmolecules coding for relatively large proteins or regulatory regions canbe synthesized by first constructing several different double-strandedmolecules that code for particular regions of the protein or regulatoryregion, followed by ligating these DNA molecules together. For example,Cunningham et al. ("Receptor and Antibody Epitopes in Human GrowthHormone Identified by Homolog-Scanning Mutagenesis," Science,243:1330-1336 (1989)), have constructed a synthetic gene encoding thehuman growth hormone gene by first constructing overlapping andcomplementary synthetic oligonucleotides and ligating these fragmentstogether. See also, Ferretfi, et al. (Proc. Nat. Acad. Sci. 82:599-603(1986)), wherein synthesis of a 1057 base pair synthetic bovinerhodopsin gene from synthetic oligonucleotides is disclosed. Once theappropriate DNA molecule is synthesized, this DNA can be cloneddownstream of a promoter. Techniques such as this are routine in the artand are well documented.

An example of another method of obtaining a nucleic acid encoding a37-kDa surface adhesin A protein is to isolate that nucleic acid fromthe organism in which it is found and clone it in an appropriate vector.For example, a DNA or cDNA library can be constructed and screened forthe presence of the nucleic acid of interest. The probe used to screenthe library can be designed to be selective for the 6B serotype protein.Methods of constructing and screening such libraries are well known inthe art and kits for performing the construction and screening steps arecommercially available (for example, Stratagene Cloning Systems, LaJolla, Calif.). Once isolated, the nucleic acid can be directly clonedinto an appropriate vector, or if necessary, be modified to facilitatethe subsequent cloning steps. Such modification steps are routine, anexample of which is the addition of oligonucleotide linkers whichcontain restriction sites to the termini of the nucleic acid. Generalmethods are set forth in Sambrook et al., "Molecular Cloning, aLaboratory Manual," Cold Spring Harbor Laboratory Press (1989).

Yet another example of a method of obtaining a Streptococcal 37-kDasurface adhesin A encoding nucleic acid is to amplify the nucleic acidfrom the nucleic acids found within the host organism. Amplificationprocedures are well known to those skilled in the art, for example seeInnis et al. "PCR Protocols: A Guide to Methods and Applications"Academic Press, Inc. 1990. An example of amplification of a nucleic acidencoding the 37-kDa protein of Streptococcus pneumoniae serotype 6B isdiscussed in the Example contained herein.

37-kDa Protein

The present invention also provides a purified polypeptide as set forthin the Sequence Listing a SEQ ID NO:2 and a purified polypeptide encodedby a nucleic acid comprising a unique fragment of at least 10nucleotides of SEQ ID NO:1. The protein can be used as a vaccinecomponent as well as a reagent for identifying host antibodies raisedagainst Streptococcus pneumoniae during infection. The purified proteincan also be used in methods for detecting the presence of Streptococcuspneumoniae.

Unique fragments of the 37-kDa protein can be identified in the samemanner as that used to identify unique nucleic acids. For example, asequence of 3 amino acids or more, derived from the sequence of the37-kDa protein as set forth in the Sequence Listing as SEQ ID NO:2 canbe used to search the protein sequence databases. Those that do notmatch a known sequence are therefore unique.

"Purified protein" as used herein means the protein or fragment issufficiently free of contaminants or cell components with which theprotein normally occurs to distinguish the protein from the contaminantsor cell components. It is not contemplated that "purified" necessitateshaving a preparation that is technically totally pure (homogeneous), butpurified as used herein means the protein or polypeptide fragment issufficiently separated from contaminants or cell components with whichit normally occurs to provide the protein in a state where it can beused in an assay, such as immunoprecipitation or ELISA. For example, the"purified" protein can be in an electrophoretic gel.

Once a nucleic acid encoding a 37-kDa pneumococcal surface adhesinprotein of serotype 6B, or a fragment of that nucleic acid, isconstructed, modified, or isolated, that nucleic acid can then be clonedinto an appropriate vector, which can direct the in vivo or in vitrosynthesis of that 37-kDa pneumococcal surface adhesin protein, orfragment thereof. The vector is contemplated to have the necessaryfunctional elements that direct and regulate transcription of theinserted gene, or gene fragment. These functional elements include, butare not limited to, a promoter, regions upstream or downstream of thepromoter, such as enhancers that may regulate the transcriptionalactivity of the promoter, an origin of replication, appropriaterestriction sites to facilitate cloning of inserts adjacent to thepromoter, antibiotic resistance genes or other markers which can serveto select for cells containing the vector or the vector containing theinsert, RNA splice junctions, a transcription termination region, or anyother region which may serve to facilitate the expression of theinserted gene or gene fragment. (See generally, Sambrook et al.).

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, apromoter system from phage lambda, or other phage promoters such as T4or T7 promoters. The promoters will typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences for example, for initiating and completing transcription andtranslation. If necessary, an amino terminal methionine can be providedby insertion of a Met codon 5' and in-frame with the downstream nucleicacid insert. Also, the carboxy-terminal extension of the nucleic acidinsert can be removed using standard oligonucleotide mutagenesisprocedures.

Alternatively, viral expression systems can be used to express thenucleic acid of the present invention, or fragments thereof For example,vaccinia virus vectors can accept large inserts and can be used toexpress foreign genes for vaccination purposes. (See, e.g., Friedman, T.Science 244:1275 (1989)). Other viral expression systems, such as thebaculovirus expression system, are also commonly used in the art.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF"-I gene) isroutinely used to direct protein secretion from yeast. (Brake et al.,"α-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae. " Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, additionof lipid moieties, and secretion of active protein. Vectors useful forthe expression of active proteins in mammalian cells are characterizedby insertion of the protein coding sequence between a strong viralpromoter and a polyadenylation signal. The vectors can contain genesconferring hygromycin resistance, gentamicin resistance, or other genesor phenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation, transduction, andelectroporation are commonly utilized for prokaryotic cells, whereascalcium phosphate, DEAE dextran, or lipofectin mediated transfection orelectroporation may be used for other cellular hosts.

Alternative vectors for the expression of genes in mammalian cells,those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VIII,hepatitis B virus surface antigen, protease Nexinl, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

Expression of the gene or hybrid gene can be by either in vivo or invitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector.Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of MRNA. In such in vitro transcription systems, thenucleic acid encoding the 37-kDa pneumococcal surface adhesin proteinwould be cloned into an expression vector adjacent to a transcriptionpromoter. For example, the Bluescript II cloning and expression vectorscontain multiple cloning sites which are flanked by strong prokaryotictranscription promoters. (Stratagene Cloning Systems, La Jolla, Calif.).Kits are available which contain all the necessary reagents for in vitrosynthesis of an RNA from a DNA template such as the Bluescript vectors.(Stratagene Cloning Systems, La Jolla, Calif.). RNA produced in vitro bya system such as this can then be translated in vitro to produce thedesired 37-kDa pneumococcal surface adhesin protein. (Stratagene CloningSystems, La Jolla, Calif.).

Another method of producing a 37-kDa pneumococcal surface adhesinprotein is to link two peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to a 37-kDa pneumococcal surfaceadhesin protein can be synthesized by standard chemical reactions,either continuous synthesis or step-wise synthesis. For example, apartial polypeptide can be synthesized and not cleaved from itssynthesis resin whereas another fragment can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform a 37-kDa pneumococcal surface adhesin protein. (Grant G. A.,"Synthetic Peptides: A User Guide," W. H. Freeman and Co., N.Y. (1992)and Bodansky, M and Trost, B., Ed., "Principles of Peptide Synthesis,"Springer-Verlag Inc., N.Y. (1993)). Alternatively, the 37-kDapneumococcal surface adhesin protein can by independently synthesized invivo as described above. Once isolated, these independent polypeptidesmay be linked to form a 37-kcDa pneumococcal surface adhesin protein viasimilar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentscan allow relatively short peptide fragments to be joined to producelarger peptide fragments, polypeptides or whole protein domains(Abrahmsen et al., Biochemistry, 30:4151 (1991)). Alternatively, nativechemical ligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al,"Synthesis of Proteins by Native Chemical Ligation," Science,266:776-779 (1994)). The first step is the chemoselective reaction of anunprotected synthetic peptide-α-thioester with another unprotectedpeptide segment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis et al., FEBSLett., 307:97 (1987), Clark-Lewis et al., J. Biol. Chem., 269:16075(1994), Clark-Lewis et al, Biochem. 30:3128 (1991), and Rajarathnam etal., Biochem. 29:1689 (1994)).

Alternatively, unprotected peptide segments can be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (non-peptide) bond (Schnolzer et al.,Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton et al.,"Techniques in Protein Chemistry IV," Academic Press, New York, pp.257-267 (1992)).

The invention also provides fragments of the 37-kDa pneumococcal surfaceadhesin protein. The polypeptide fragments of the present invention canbe recombinant proteins obtained by cloning nucleic acids encodingfragments of the polypeptide in an expression system capable ofproducing the polypeptide fragments thereof, as described above for the37-kDa protein. For example, one can determine an immunoreactive regionof a 37-kDa pneumococcal surface adhesin protein which can cause asignificant immune response, clone the nucleic acid encoding thatpolypeptide into an expression vector, and isolate that particularpolypeptide for further uses, such as diagnostics, therapy, andvaccination. In one example, amino acids found to not contribute to theimmunoreactivity and/or specificity can be deleted without a loss in therespective activity.

For example, amino or carboxy-terminal amino acids, can be sequentiallyremoved from the 37-kDa pneumococcal surface adhesin protein and theimmunoreactivity tested in one of many available assays. Alternatively,internal amino acids can be sequentially removed and theimmunoreactivity tested for each of the deletions. In another example, afragment of a 37-kDa pneumococcal surface adhesin protein can comprise amodified polypeptide wherein at least one amino acid has beensubstituted for the naturally occurring amino acid at specificpositions, or a portion of either amino terminal or carboxy terminalamino acids, or even an internal region of the polypeptide, can bereplaced with a polypeptide fragment or other moiety, such as biotin,which can facilitate in the purification of the modified 37-kDapneumococcal surface adhesin protein. For example, a modified 37-kDapneumococcal surface adhesin protein can be fused to a maltose bindingprotein, through either peptide chemistry of cloning the respectivenucleic acids encoding the two polypeptide fragments into an expressionvector such that the expression of the coding region results in a hybridpolypeptide. The hybrid polypeptide can be affinity purified by passingit over an amylose ainity column, and the modified 37-kDa pneumococcalsurface adhesin protein can then be separated from the maltose bindingregion by cleaving the hybrid polypeptide with the specific proteasefactor Xa. (See, e.g., New England Biolabs Product Catalog, 1996, pg.164.)

Immunoreactive fragments of a 37-kDa pneumococcal surface adhesinprotein can also be synthesized directly or obtained by chemical ormechanical disruption of larger 37-kDa pneumococcal surface adhesinprotein. An immunoreactive fragment is defined as an amino acid sequenceof at least about 6 consecutive amino acids derived from the naturallyoccurring amino acid sequence, which has the relevant activity, e.g.,evoking an immune response.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the immunoreactivity of the peptide is not significantlyimpaired compared to the 37-kDa pneumococcal surface adhesin protein.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, etc. In any case, the peptide must possess a bioactiveproperty, such as immunoreactivity. Functional or active regions of the37-kDa pneumococcal surface adhesin protein may be identified bymutagenesis of a specific region of the protein, followed by expressionand testing of the expressed polypeptide. Such methods are readilyapparent to a skilled practitioner in the art and can includesite-specific mutagenesis of the nucleic acid encoding the receptor.(See, e.g., Smith, M "In vitro mutagenesis" Ann. Rev. Gen., 19:423-462(1985) and Zoller, M. J. "New molecular biology methods for proteinengineering" Curr. Opin. Struct. Biol., 1:605-610 (1991)).

Antibodies

The present invention also provides a purified antibody whichselectively binds with the polypeptide encoded by the nucleic acid setforth in the sequence listing as SEQ ID NO:1, or a polypeptide encodedby a unique fragment of at least 10 nucleotides of SEQ ID NO:1. Theantibody (either polyclonal or monoclonal) can be raised to the 37-kDapneumococcal surface adhesin protein of a unique fragment thereof, inits naturally occurring form and in its recombinant form. The antibodycan be used in techniques or procedures such as diagnostics, treatment,or vaccination.

Antibodies can be made by many well-known methods (See, e.g. Harlow andLane, "Antibodies; A Laboratory Manual" Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can beinjected into an animal in an amount and in intervals sufficient toelicit an immune response. Antibodies can either be purified directly,or spleen cells can be obtained from the animal. The cells can thenfused with an immortal cell line and screened for antibody secretion.The antibodies can be used to screen nucleic acid clone libraries forcells secreting the antigen. Those positive clones can then besequenced. (See, for example, Kelly et al., Bio/Technology, 10:163-167(1992); Bebbington et al., Biol/Technology, 10:169-175 (1992)).

The phrase "selectively binds" with the polypeptide refers to a bindingreaction which is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bound to aparticular protein do not bind in a significant amount to other proteinspresent in the sample. Selective binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. A variety of immunoassay formats may be usedto select antibodies selectively bind with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein. See Harlow andLane "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications,New York, (1988), for a description of immunoassay formats andconditions that could be used to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, "Basic and ClinicalImmunology," (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane("Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, NewYork, (1988)).

The present invention also provides a monoclonal antibody designated1E7A3D7C2, or a fragment thereof which retains the characteristics ofantibody 1E7A3D7C2, such as its binding specificity and its bindingaffinity.

The present invention also provides a monoclonal antibody designated1B6E12H9, or a fragment thereof which retains the characteristics ofantibody 1B6E12H9.

The present invention also provides a monoclonal antibody designated3C4D5C7, or a fragment thereof which retains the characteristics ofantibody 3C4D5C7.

The present invention also provides a monoclonal antibody designated4E9G9D3, or a fragment thereof which retains the characteristics ofantibody 4E9G9D3.

The present invention also provides a monoclonal antibody designated4H5C10F3, or a fragment thereof which retains the characteristics ofantibody 4H5C10F3.

The present invention also provides a monoclonal antibody designated6F6F9C8, or a fragment thereof which retains the characteristics ofantibody 6F6F9C8.

The present invention also provides a monoclonal antibody designated8G12G11B10, or a fragment thereof which retains the characteristics ofantibody 8G12G11B10.

Vaccines

Also provided by the present invention is a vaccine comprising animmunogenic polypeptide encoded by the nucleic acid as set forth in theSequence Listing as SEQ ID NO:1, or a unique fragment of at least 10nucleotides of SEQ ID NO:1. The polypeptides provided by the presentinvention can be used to vaccinate a subject for protection from aparticular disease, infection, or condition caused by the organism fromwhich the 37-kDa pneumococcal surface adhesin protein of a uniquefragment thereof was derived.

Polypeptides of a 37-kDa pneumococcal surface adhesin protein ofserotype 6B or a unique fragment thereof, therefore, can be used toinoculate a host organism such that the host generates an active immuneresponse to the presence of the polypeptide or polypeptide fragmentwhich can later protect the host from infection by organism from whichthe polypeptide was derived. One skilled in the art will appreciate thatan immune response, especially a cell-mediated immune response, to a37-kDa pneumococcal surface adhesin protein from a specific strain canprovide later protection from reinfection or from infection from aclosely related strain. The 37-kDa protein provided by the presentinvention, however, is relatively conserved among many of the variousserotypes of S. pneumoniae and can serve as a multivalent vaccine.

Immunization with the 37-kDa pneumococcal surface adhesin protein can beachieved through artificial vaccination. (Kuby, J. "Immunology" W. H.Freeman and Co. New York, 1992). This immunization may be achieved byadministering to subjects the 37-kDa pneumococcal surface adhesinprotein either alone or with a pharmaceutically acceptable carrier.

Immunogenic amounts of the 37-kDa pneumococcal surface adhesin proteincan be determined using standard procedures. Briefly, variousconcentrations of the present polypeptide are prepared, administered tosubjects, and the immunogenic response (e.g., the production ofantibodies to the polypeptide or cell mediated immunity) to eachconcentration is determined. Techniques for monitoring the immunogenicresponse, both cellular and humoral, of patients after inoculation withthe polypeptide, are very well known in the art. For example, samplescan be assayed using enzyme-linked immunosorbent assays (ELISA) todetect the presence of specific antibodies, such as serum IgA (Hjelt etal. J. Med. Virol. 21:39-47, (1987)), or lymphocytes or cytokineproduction can be monitored. The specificity of a putative immunogenicantigen of any particular polypeptide can be ascertained by testingsera, other fluids or lymphocytes from the inoculated patient forcross-reactivity with other closely related 37-kDa pneumococcal surfaceadhesin proteins.

The amount of a polypeptide of the 37-kDa pneumococcal surface adhesinprotein administered will depend on the subject, the condition of thesubject, the size of the subject, etc., but will be at least animmunogenic amount. The polypeptide can be formulated with adjuvants andwith additional compounds, including cytokines, with a pharmaceuticallyacceptable carrier.

It is also contemplated that immunization against Streptococcuspneumoniae can be achieved by a "naked" DNA vaccine approach. Briefly,DNA constructs containing promoter sequences upstream of the 37-kDaprotein or specific antigen coding sequences can be injected into muscletissue or administered via the mucosa and result in expression of viralantigens that induce a protective immune response.

The pharmaceutically acceptable carrier or adjuvant in the vaccine ofthe present invention can be selected by standard criteria (Arnon, R.(Ed.) "Synthetic Vaccines" I:83-92, CRC Press, Inc. Boca Raton, Fla.,1987). By "pharmaceutically acceptable" is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected compound withoutcausing any undesirable biological effects or interacting in aundesirable manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier oradjuvant may depend on the method of administration and the particularpatient.

Methods of administration can be by oral, sublingual, mucosal, inhaled,absorbed, or by injection. Actual methods of preparing the appropriatedosage forms are known, or will be apparent, to those skilled in thisart; for example, see Remington's Pharmaceutical Sciences (Martin, E. W.(ed.) latest edition Mack Publishing Co., Easton, Pa.

Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

Detection Methods

The present invention also provides a method of detecting the presenceof the Streptococcus pneumoniae in a sample, comprising the steps ofcontacting a sample suspected of containing Streptococcus pneumoniaewith nucleic acid primers capable of hybridizing to a nucleic acidcomprising a unique portion of the nucleic acid set forth in theSequencing Listing as SEQ ID NO:1, amplifying the nucleic acid,detecting the presence of an amplification product, the presence of theamplification product indicating the presence of Streptococcuspneumoniae in the sample. Alternatively, a unique fragment of thenucleic acid of SEQ ID NO:1 can be used to specifically identify anon-selectively amplified nucleic acid.

The specific amplification methods are well known in the art. Forexample, and as disclosed in the Example contained herein the polymerasechain reaction (PCR) can be used to amplify nucleic acid in a samplespecific for Streptococcus pneumoniae. Other amplification techniquescan also be used to detect the presence of Streptococcus pneumoniae in asample, such as the ligase chain reaction (LCR), the self-sustainedsequence replication (3SR) system, the transcription-based amplificationsystem (TAS), and the RNA replication system based on Qβ replicase.

The amplified nucleic acid can be detected in any number of detectionassays. For example, the primers can be radio-labeled such that theamplification product containing these primers can be detected by thedetecting the radioactive decay from those primers. Alternatively, theprimers can contain other detectable moieties, such as biotin, or theamplified nucleic acid can be stained and visualized, such as withethidium bromide staining.

The present invention also provides a method of detecting the presenceof Streptococcus pneumoniae in a subject, comprising the steps ofcontacting an antibody-containing sample from the subject with purifiedpolypeptide encoded by the nucleic acid set forth in the SequenceListing as SEQ ID NO:1, or a purified polypeptide encoded by a nucleicacid comprising a unique fragment of at least 10 nucleotides of SEQ IDNO:1, and detecting the binding of the antibody with the polypeptide,the binding indicating the presence of Streptococcus pneumoniae in thesubject.

The present invention further provides a method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof contacting a sample from the subject with an antibody whichselectively binds the purified polypeptide encoded by the nucleic acidset forth in the Sequence Listing as SEQ ID NO:1, or a purifiedpolypeptide encoded by a nucleic acid comprising a unique fragment of atleast 10 nucleotides of SEQ ID NO:1 and detecting the binding of theantibody with an antigen, the binding indicating the presence ofStreptococcus pneumoniae in the subject.

There are numerous immunodiagnostic methods that can be used to detectantigen or antibody as the following non-inclusive examples illustrate.These methods, as well as others, can not only detect the presence ofantigen or antibody, but quantitate antigen or antibody as well.

Immunoassays such as immunofluorescence assays (IFA), enzyme linkedimmunosorbent assays (ELISA) and immunoblotting can be readily adaptedto accomplish the detection and quantitation of the antigen or antibody.An ELISA method effective for the detection of the antigen, for example,can be as follows: (1) bind the antibody to a substrate; (2) contact thebound antibody with a fluid or tissue sample containing the antigen; (3)contact the above with a secondary antibody bound to a detectable moiety(e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme);(4) contact the above with the substrate for the enzyme; (5) contact theabove with a color reagent; (6) observe color change. The above methodcan be readily modified to detect antibody as well as antigen.

Another immunologic technique that can be useful in the detectionutilizes monoclonal antibodies (MAbs) for detection of antibodies thatspecifically bind a specific antigen. Briefly, sera or other body fluidfrom the subject is reacted with the antigen bound to a substrate (e.g.an ELISA 96-well plate). Excess sera is thoroughly washed away. Alabeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonalantibody is then reacted with the previously reacted antigen-serumantibody complex. The amount of inhibition of monoclonal antibodybinding is measured relative to a control (no patient serum antibody).The degree of monoclonal antibody inhibition can be a specific test fora particular species or subspecies or variety or strain since it isbased on monoclonal antibody binding specificity. MAbs can also be usedfor detection directly in cells by IFA.

A micro-agglutination test can also be used to detect the presence ofantibodies in a subject. Briefly, latex beads (or red blood cells) arecoated with the antigen and mixed with a sample from the subject, suchthat antibodies in the tissue or body fluids that are specificallyreactive with the antigen crosslink with the antigen, causingagglutination. The agglutinated antigen-antibody complexes form aprecipitate, visible with the naked eye or detectable by aspectrophotometer. In a modification of the above test, antibodiesspecifically reactive with the antigen can be bound to the beads andantigen in the tissue or body fluid thereby detected.

In addition, as in a typical sandwich assay, the antibody can be boundto a substrate and contacted with the antigen. Thereafter, a labeledsecondary antibody is bound to epitopes not recognized by the firstantibody and the secondary antibody is detected.

In the diagnostic methods taught herein, the antigen can be bound to asubstrate and contacted by a fluid sample such as serum, urine, salivaor gastric juice. This sample can be taken directly from the subject, orin a partially purified form. In this manner, antibodies specific forthe antigen (the primary antibody) will specifically bind with the boundantigen. Thereafter, a secondary antibody bound to, or labeled with, adetectable moiety can be added to enhance the detection of the primaryantibody. Generally, the secondary antibody or other ligand which bindsspecifically with a different epitope of the antigen or nonspecificallywith the ligand or bound antibody, will be selected for its ability tobind with multiple sites on the primary antibody. Thus, for example,several molecules of the secondary antibody can bind with each primaryantibody, making the primary antibody more detectable.

The detectable moiety will allow visual detection of a precipitate or acolor change, visual detection by microscopy, or automated detection byspectrometry, radiometric measurement or the like. Examples ofdetectable moieties include fluorescein and rhodamine (for fluorescencemicroscopy), horseradish peroxidase (for either light or electronmicroscopy and biochemical detection), biotin-streptavidin (for light orelectron microscopy) and alkaline phosphatase (for biochemical detectionby color change). The detection methods and moieties used can beselected, for example, from the list above or other suitable examples bythe standard criteria applied to such selections (Harlow et al.,"Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., (1988)).

Methods of Treating and Preventing Infection

The present invention also provides a method of preventing Streptococcuspneumoniae infection in a subject, comprising administering to thesubject a prophylactically effective amount of a vaccine comprising animmunogenic polypeptide encoded by the nucleic acid encoding the 37-kDaprotein of Streptococcus pneumoniae as set forth in the Sequence Listingas SEQ ID NO:1, or an immunogenic polypeptide encoded by a nucleic acidcomprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1.,either alone or with a pharmaceutically acceptable carrier.

The present invention further provides a method of preventingStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a prophylactically effective amount of ananti-idiotype antibody to the polypeptide encoded by the nucleic acid asset forth in the Sequence Listing as SEQ ID NO:1, or a polypeptideencoded by a nucleic acid comprising a unique fragment of at least 10nucleotides of SEQ ID NO:1, either alone or with a pharmaceuticallyacceptable carrier.

Anti-idiotype antibodies represent the image of the original antigen andcan serve as a vaccine to induce an immune response to a pathogenicantigen, therefore avoiding immunization with the pathogen itself. Thistype of protection has been demonstrated by immunizing mice withanti-idiotype antibody to the binding site of TEPC-15, the majorcomponent of the pneumococcal cell wall C polysaccharide. Mice immunizedwith these anti-idiotype antibodies were immune when they were laterchallenged with live pneumococci. Mice have also been used todemonstrate anti-idiotype antibodies can provide protection againsthepatitis B virus, rabies virus, Sendai virus, Streptococcus pneumoniae,Listeria monocytogenes, Trypanosoma rhodesiense, and Schistosomamansoni. (See, Kuby, J. "Immunology" W. H. Freeman and Co. New York,1992).

The present invention further provides a method of treating aStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a therapeutically effective amount of anantibody to the polypeptide encoded by the nucleic acid as set forth inthe Sequence Listing as SEQ ID NO:1, or a polypeptide encoded by anucleic acid comprising a unique fragment of at least 10 nucleotides ofSEQ ID NO:1, either alone or with a pharmaceutically acceptable carrier.

Treating a subject already infected with a particular organism byadministering to the subject antibody against the organism is well knownin the art. For example, immune globulin isolated from animals or humanspreviously exposed to rabies virus is currently a therapy for rabiesvirus infection. Better treatment of infected individuals can beachieved by administering to those individuals monoclonal antibodiessince those monoclonals react or bind more specifically that thepolyclonals. (See, e.g. Kaplan et al. "Rabies" Sci. Am. 242:120-134(1980)).

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how theattenuated prokaryotes claimed herein are made and evaluated, anddemonstrates the methods of the present invention, and is intended to bepurely exemplary of the invention and is not intended to limit the scopeof what the inventors regard as their invention. Efforts have been madeto ensure accuracy with respect to numbers (e.g., amounts, temperature,etc.) but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in °C.and pressure is at or near atmospheric.

EXAMPLES

Bacterial strains

The S. pneumoniae strain R36A was kindly provided by D. E. Briles(University of Alabama at Birmingham). Twenty-four serotypes of S.pneumoniae were provided by R. Facklam, Centers for Disease Control(CDC), Atlanta, Ga. These serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8,9N, 9V, 10A, 11F, 11A, 12F, 14, 15B, 18C, 19A, 19F; 20, 22F, 23F, and33F. Entex,ococcus avium, E. casseliflavus, and E. gallinarum were alsoprovided by R. Facklam. Anaerobic bacteria were obtained from V. R.Dowell, CDC. These included Bacteroides asaccharolyticus, B. fragilis,B. intermedius, B. thetaiotaomicron, Eubacterium lentum, Fusobacteriumnecrophorum, F. nucleatum, Peptostreptococcus anaerobius, P.asaccharolyticus, Propionibacterium acnes, and Staphylococcussaccharolyticus. Branhamella catarrhalis and Bordetella parapertussiswere obtained from R. Weaver, CDC. Mycobacterium tuberculosis wasprovided by R. C. Good, CDC. R. Barnes, CDC, provided Chlamydiapneumoniae. The following remaining bacteria were from the stockcollection of the Immunology Laboratory, CDC: Bordetella pertussis,Enterobacter aerogenes, E. agglomerans, E. cloacae, E. gergoviae,Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae (typesa-f), Legionella micdadei, L. pneumophila, Mycoplasma pneumoniae,Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus,Streptococcus agalactiae, S. equisimilis, S. pyogenes, and group Gstreptococci.

Production of MAbs

Female BALB/c mice were immunized with whole cell suspensions of S.pneumoniae R36A, a rough derivative of the capsular type 2 strain D39(Avery et al. (1944) J. Exp. Med. 79:137-157). The mice were immunizedby intravenous injection three times and intraperitoneal injection onetime. The maximum number of cells injected at any time was 10⁸. Fusionwas done on day 25 by using standard procedures (Clafin et al. (1978)Curr. Top. Microbiol. Immunol. 81:107-109). Spleen cells of 4 mice werefused with Sp2/0-Ag14 myeloma cells (Schulman et al. (1978) Nature(London) 276:269-270). Culture fluids of the growing hybridomas weretested for antibodies to S. pneumoniae whole cells in an ELISA. A clonedesignated 1E7A3D7C2 was one of 10 selected for further study.

ELISA

Screening of hybridoma culture supernatants was done by ELISA. U-bottommicrotitration plates (Costar, Cambridge, Mass.) were sensitized with 50μl of S. pneumoniae whole cell suspension (10⁹ CFU/ml) diluted 1:4,000in 0.1M carbonate buffer, pH 9.6, and kept for 16 h at 4° C. The plateswere washed 5 times with 0.9% NaCl containing 0.05% Tween 20 (NaCl-T).Culture supernatants (50 μl) from the fusion plates were added to 50 μlof a solution containing 2% bovine serum albumin (BSA), 10% normalrabbit serum, 0.3% Tween-20, and 0.02% Merthiolate in phosphate bufferedsaline (PBS), pH 7.2, (ELISA diluent) (Wells. et al. (1987) J. Clin.Microbiol. 25:516-521) in the plates and were incubated for 30 min at37° C. The plates were washed 5 times with NaCl-T. Fifty microliters ofgoat anti-mouse immunoglobulin horseradish peroxidase conjugate, dilutedin ELISA diluent was added to each well. The plates were incubated for30 min at 370° C. The plates were washed, and 50 μl of3,3',5,5'-tetramethylbenzidine (0.1 mg/ml in 0.1M sodium acetate, 0.1Mcitric acid (pH 5.7! with 0.005% hydrogen peroxide) was added to eachwell and incubated for 30 min at 37° C. The reaction was stopped byadding 1 ml of 4M H₂ SO₄ and the optical density was read on a DynatechELISA Reader (Dynatech Laboratories, Inc., Alexandria, Va.) at 450 nm.An optical density of >0.200 was considered positive.

SDS-PAGE and immunoblot analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed by the method of Tsang et al. (Tsang et al. (1983) MethodsEnzymol. 92:377-391), using an 8% acrylamide resolving gel. Equalvolumes of sample buffer (5% SDS-10% 2-mercaptoethanol-20% glycerol in0.01M Tris HCL, pH 8.0!) and cell suspension containing 2.4 μg proteinper μl were mixed, heated at 100° C. for 5 min, and a 5-μl portion wasapplied to 1 of 15 wells. If the final protein content of the portion ofsample to be tested was <1.2 μg/μl, a volume up to 10 μl of sample wasapplied to achieve a final concentration of 6 μl of protein per well.Protein concentrations were determined by the method of Markwell et al.(Markwell et al. (1978) Anal. Biochem. 87:206-210), with BSA as thestandard.

Proteins separated by SDS-PAGE were either silver stained by the methodof Morrissey (Morrissey, J. H. (1981) Anal. Biochem. 117:307-310) orelectroblotted onto nitrocellulose (Schleicher & Schnell, Inc., Keene,N. H.). The immunoblot procedure was done according to the method ofTsang et al. (Tsang et al. (1983) Methods Enzymol. 92:377-391) withslight modifications. The blots were given three 5-min washes with PBS,pH 7.2, containing 0.3% Tween-20 and were gently agitated overnight (16h) at 25° C. The blots were blocked for 1 h with casein-thimerosalbuffer (CTB) (Kenna et al. (1985) J. Immunol. Meth. 85:409-419). Afterthree rinses with CTB, the blots were exposed to goat anti-mouseimmunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories,Richmond, Calif.) for 2 h at 25° C. Conjugate dilutions (1:2,000) weremade in CTB. The blots were again rinsed three times with CTB andexposed to 3-3'diaminobenzadine-4-hydrochloride in PBS, pH 7.2 (0.5mg/ml), with 0.003% H₂ O₂ for 5 min at 25° C. Reactivity was expressedas a visible colored band on the nitrocellulose paper. Lowmolecular-mass protein standards (Bio-Rad) were used in PAGE andimmunoblotting. Rabbit antisera to the protein standards were used todevelop the standards (Carlone, G. M. (1986) Anal. Biochem. 155:89-91).Molecular masses were calculated by the method of Neville and Glossman(Neville et al. (1974) Methods Enzymol. 32:92-102) using appropriatemolecular mass standards.

IFA

A bacterial suspension containing approximately 400-500 CFU per field(10 μl) was allowed to dry at room temperature on each well ofacetone-resistant, 12-well (5 mm diameter), glass slides (25×75 mm)(Cel-Line Associates, Newfield, N.J.). The slides were then immersed inacetone for 10 min and air dried at room temperature. MAbs were added tothe slides, which were incubated for 30 min at 37° C. After incubation,the slides were gently rinsed with PBS and soaked twice at 5-minintervals, blotted on filter paper, and air dried at room temperature.Fluorescein-labeled rabbit anti-mouse immunoglobulin (courtesy of W. F.Bibb, CDC) was then added, and the slides were incubated for 30 min at37° C. They were then washed twice with PBS and gently blotted on filterpaper. Slides were covered with carbonate-buffered mounting fluid, pH9.0, and cover slips and were then read with a Leitz Dialux 20fluorescence microscope equipped with a HBO-100 mercury incident lightsource, an I cube filter system, a 40× dry objective lens, and 6.3×binoculars (E. Leitz, Inc., Rockleigh, N.J).

Immunoelectron-microscopy

Pneumococcal cells were washed two times with PBS and fixed in a mixtureof 1% paraformaldehyde-0.1% glutaraldehyde (freshly made) for 20 min at4° C. The cells were dehydrated in a graded alcohol series and then in a1:1 mixture of absolute ethanol and Lowicryl K4M (Ladd ResearchIndustries, Inc., Burlington, Vt.) for 1 h at 4° C. The cells werepelleted and suspended in a 1:2 mixture of absolute ethanol and LowicrylK4M for 1 h at 4° C. They were again pelleted and suspended in LowicrylK4M (undiluted) for 16 h at 4° C.

The cells were transferred to fresh Lowicryl K4M two times during thenext 24-hour period. The Lowicryl K4M-treated cells were imbedded ingelatin capsules, which were placed inside a box lined with aluminumfoil. The capsules were hardened by holding them in the box 35 cm from ashort-wave UV light source for 72 h at -20° C. The box was brought toroom temperature, and the capsules were allowed to continue hardeningfor up to 14 days.

Samples of the capsule were cut into 100-μm thin sections and picked upon nickel grids. Grids containing the sample were placed on a droplet ofovalbumin solution in PBS containing sodium azide (E. Y. Laboratories,Inc., San Mateo, Calif.) for 5 min. The grids (wet) were transferred toa solution of primary MAbs diluted in a solution of BSA reagent (1% BSAin PBS containing 0.1% Triton X-100, Tween 20, and sodium azide) (E. Y.Laboratories) and incubated for 1 h at room temperature or 18 to 48 h at4° C. in a moist chamber. For antibody binding controls, other gridswere wetted with MAbs against Legionella pneumophila. The grids wererinsed two times with PBS and incubated on droplets of goat anti-mouseIgG-labeled colloidal gold particles (20 μm)(E. Y. Laboratories) for 1 hat room temperature. The grids were rinsed two times and post-stainedwith osmium tetroxide, uranyl acetate, and lead citrate. The grids wereexamined with a Philips 410 transmission electron microscope.

CBA/CaBN/J Mice

X-linked immune deficiency (xid) of CBA/N mice as prepared by Wicker, L.S. and I. Seher, Curr. Top. Microbiol. Immunol. 124:86-101 were used tostudy the protection afforded by the 37 kDa protein.

Example 1 Monoclonal Antibodies

Hybridoma clone 1E7A3D7C2 produced MAbs that reacted with a37-kilodalton (kDa) protein antigen (pneumococcal fimbrial protein A)found in S. pneumoniae. The MAbs reacted with an antigen fractionated inSDS-PAGE, yielding a single immunoblot band. This indicates that the MAbreacted with epitopes found only on the 37-kDa antigen (pneumococcalfimbrial protein A). The MAbs produced by the immunization of mice withpneumococcal cells reacted with all pneumococcal strains tested (24serotypes) to yield a sensitivity of 100%. For specificity, 55 differentnonpneumococcal strains of bacteria that can also cause respiratoryinfections (Donowitz et aL (1985) In: Principles and practices ininfectious diseases, 2nd ed. (G. L. Mandell, R. G. Douglas, and J. E.Bennett, ed.) John Wiley & Sons, Inc., New York, pp.394-404) were testedfor antigens reacting with the MAbs. The latter strains represented 19genera and 36 species of bacteria. None of the strains tested reactedwith the pneumococcal MAbs, thus yielding a specificity of 100%.

Of 44 patients known to have pneumococcus disease, 34 (77%) hadantibodies that reacted with the 37-kDa antigen (pneumococcal fimbrialprotein A) by Western immunoblot.

The MAbs reacted with whole pneumococcal cells to yield a positive testresult in both the ELISA and IFA. Results from both the ELISA and theIFA indicate that the antigen has exposed epitopes on the surface of thecell or that the immunoglobulin and other immunologic reagents are ableto penetrate the pneumococcal cell walls.

Several strains of group A streptococci were tested forimmunofluorescence after reacting with the pneumococcus MAbs. None ofthe heterologous bacterial cells fluoresced in this test, indicatingthat the IFA reaction was specific for pneumococcus cells.

To further determine the location on the cell of the 37-kDa antigen(pneumococcal fimbrial protein A) epitopes reacting with the MAbs,immunolabeling experiments were performed. The cells were typical ofgram-positive cocci in the process of division. A large portion of theantigen appears to be intracellular since there is no coating orlayering of the labeled MAbs around the cell. The large patch ofcolloidal gold staining indicates that the MAbs bound antigen locatedinside the cell wall. There was no colloidal gold binding to controlpneumococci that were exposed to the MAbs against L. pneumophila.

Example 2

Cloning of the Pneumococcal Fimbrial Protein A Gene

Streptococcus pneumoniae DNA digested with restriction enzyme Sau3A1 wasligated to BamHi digested pUC13 and transformed into E. coli TB1.Recombinant clones were identified by colony immunoblot using the 37-kDamonoclonal antibody. The plasmid pSTR3-1 is an example of thepneumococcal-fimbrial protein A gene cloned into pUC13 .

Example 3

Preparation of Purified 37 kDa Protein Antigen

Two methods for preparing the 37 kDa protein are used. (1) Streptococcuspneumoniae is conventionally cultured and the cells harvested. Purified37 kDa protein antigen (pneumococcal fimbrial protein A) is isolatedfrom the Streptococcus pneumoniae cell mass by extraction with anon-ionic detergent and further purified by ammonium sulfatefractionation and isoelectric focusing. (2) E. coli TB1 strainscontaining plasmid pSTR3-1 is cultured conventionally and the cellsharvested. For improved yields, E. coli strains, transformed with anexpression vector that carries a strong, regulated prokaryotic promoterand which contains the gene coding for the 37 kDa protein, is used.Suitable expression vectors are those that contain a bacteriophage λPLPromoter (e.g., pKK1773-3), a hybrid trp-lac promoter (e.g., pET-3a) ora bacteriophage T7 promoter. The 37 kDa protein (PfpA) is then extractedfrom the separated cell mass.

PROTECTION EXPERIMENTS WITH 37 kDa PROTEIN

Experiment No. 1

Twenty CBA/CaHN/J mice carrying the xid (x-linked immunodeficiency)mutation were used in this protection study. They were tested forprotection against challenge with a virulent type 3 Streptococcuspneumoniae strain, WU2. Mice were anesthetized with Ketamine/Rompun andbled infraorbitally to obtain pre-immunization sera. 37-kDa protein(pneumococcal fimbrial protein A) was emulsified in complete Freund'sadjuvant (CFA) to a protein concentration of 54 μg per ml. Ten mice wereinjected subcutaneously into 2 axillary and 2 inguinal sites at 0.1 mlper site, delivering approximately 22 μg protein/mouse. Ten control micewere treated identically with CFA and buffer substituting for protein.Fourteen days later, the ten test mice were injected intraperitoneally(IP) with 100 μg of the 37-kDa protein; controls were injected IP withbuffer eight days following the IP immunizations, all mice were bledinfraorbitally to obtain post-immunization sera, and challengedintravenously (IV) with 60 CFU of a log phase culture of S. pneumoniaestrain WU2, a virulent capsular type 3 strain. Mice were observed for 21days, and deaths were recorded.

Sera were collected prior to immunizations to establish baselineexposures, and also following the full immunization protocol (but beforechallenge) in order to correlate circulating antibody to the 37 kDaprotein with protection.

Days post challenge:

1--no deaths

2--3 control mice dead

3--2 control mice dead

4--2 control mice dead, one sick

5--1 control mouse dead

6-21 no deaths

Immunized with 37 kDa protein: 10/10 survived

Controls with no protein:2/10 survived (8/10 died)

Difference statistically significant: (p=0.0008) Rank sum test

Experiment No. 2

Twenty CBA/CaHN/J mice carrying the xid mutation were injected accordingto the following protocol:

1. All mice were bled prior to immunization to establish baselineimmunity. Ten test mice were immunized subcutaneously in four sites witha total of 21 μg of 37-kDa protein antigen (pneumococcal fimbrialprotein A) emulsified in Complete Freund's adjuvant (CFA). Ten controlmice were immunized identically with CFA and buffer substituting for theantigen.

2. Fourteen days later, the mice were boosted intraperitoneally (I.P.)with 100 μg of the 37 kDa protein antigen (test mice) or with buffer(controls). No adjuvant was used with this booster immunization.

3. Eight days later, all mice were bled via the infraorbital sinus andthe sera were collected and pooled into the two groups (immunized andcontrols). At the same time, blood was collected from individual mice toassay for antibody responses.

4. One day later, two additional mice were injected I.O. with 0.1 ml ofpooled immune sera to attempt to passively transfer immunity. Threeadditional mice were injected I.P. with 0.1 ml of pooled control mousesera. (only five mice were injected at this step because of the smallamount of sera obtained from the immunized mice.).

5. One hour after the I.P. injections, these five mice were challengedintravenously (I.V.) with 140 colony-forming units (CFU) of a mid-logphase pneumococcal type 3 strain, WU2.

6. At the same time, the eighteen (8 test and 10 control)* mice werechallenged I.V. with the same culture of WU2.

7. Deaths were tallied daily.

    ______________________________________                                        RESULTS:            No. Dead/No. Challenged                                   ______________________________________                                        Immunized with the 37 kDa protein:                                                                 0/8*                                                     Control mice:       10/10                                                     Passive Protection:                                                           mice receiving immune sera:                                                                       0/2                                                       Mice receiving control sera:                                                                      3/3                                                       ______________________________________                                         *Two of ten test mice died of other causes prior to challenged with WU2. 

Mice immunized with the 37 kDa protein were protected from fatalchallenge with strain WU2, and this immunity could be passivelytransferred with sera from immunized mice.

Experiment No. 3

An enzyme-linked immunosorb6nt assay (ELISA) was developed usingpurified S. pneumoniae 37-kDa protein antigen as a capture for humanantibodies. Paired sera were tested from children, less than 24 monthsof age, known to have pneumococcal pneumonia. Disease confirmation wasdetermined by blood culture or antigen in the urine. It was found that35% (9/26) had antibody titers greater than sera from non-ill childrenof the same age group, p=0.06. This illustrates that some of thechildren responded to the 37-kDa protein antigen after naturalinfection.

PREPARATION OF THE 37 kDa PROTEIN OR POLYPEPTIDE CONJUGATE

Conjugates can be prepared by use of a carrier protein bound to the37-kDa protein or polypeptides derived from the 37-kDa protein via alinker, to elicit a T cell dependent response. Such carrier proteinscould be any immunogenic protein, for example, keyhole limpethemocyanin, bovine serum albumin, tetanous toxoid, diphtheria toxoid,and bacterial outer membrane proteins. Examples of bacterial outermembrane proteins, useful as conjugates, include outer membrane proteinsof Neisseria meningitides and Haemophilus influenzae. Neisseriameningitides can be an organism selected from Neisseria meningitides,group A, B, or C.

In addition, the 37-kDa protein or polypeptides thereof can be used in aconjugate where the 37-kDa protein or polypeptides thereof are theT-cell dependent immunogenic carrier for polysaccharide antigens thatare B-cell stimulators. This is based on the theory that polysaccharideantigens are B-cell stimulators and that protective immunity is usuallygenerated by a combination of B-cell and T-cell stimulation. Proteinantigens exhibit T-cell dependent properties; i.e., booster and carrierpriming. T-cell dependent stimulation is important because children lessthan two years of age do not respond to T-cell independent antigens. Theattachment or conjugation of antigens can be accomplished byconventional processes, such as those described in U.S. Pat. No.4,808,700, involving the addition of chemicals that enable the formationof covalent chemical bonds between the carrier immunogen and theimmunogen.

In use, the 37-kDa protein antigen of this invention can be administeredto mammals; e.g., human, in a variety of ways. Exemplary methods includeparenteral (subcutaneous) administration given with a nontoxic adjuvant,such as an alum precipitate or peroral administration given afterreduction or ablation of gastric activity; or in a pharmaceutical formthat protects the antigen against inactivation by gastric juice (e.g., aprotective capsule or microsphere).

The dose and dosage regimen will depend mainly upon whether the antigenis being administered for therapeutic or prophylactic purposes, thepatient, and the patient's history. The total pharmaceutically effectiveamount of antigen administered per dose will typically be in the rangeof about 2 μg to 50 μg per patient.

For parenteral administration, the antigen will generally be formulatedin a unit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic and nontherapeutic. Examples of suchvehicles include water, saline, Ringer's solution, dextrose solution,and 5% human serum albumin. Non aqueous vehicles, such as fixed oils andethyl oleate, may also be used. Liposomes may be used as vehicles. Thevehicle may contain minor amounts of additives, such as substances whichenhance isotonicity and chemical stability; e.g., buffers andpreservatives.

Example 4

Bacterial strains. All isolates of S. pneumoniae were provided andserotyped by the Streptococcal Reference Laboratory, Division ofBacterial and Mycotic Diseases, NCID, Centers for Disease Control andPrevention (CDC). The pneumococcal serotype 6B strain used for cloningand sequencing was a CDC reference strain (SP-86).E. coli DH5α (BethesdaResearch Laboratories, Gaithersburg, Md.) was used as the recipient hostfor plasmids, pUC19 and its derivatives.

S. pneumoniae strains were grown on Trypticase soy agar plates with 5%sheep blood cells or, where indicated, in Todd-Hewitt broth containing0.5% yeast extract. E. coli cultures were grown in Luria broth which,when required, was supplemented with 100 μg/ml of ampicillin (SigmaChemical Co., St. Louis, Mo.).

Cloning and sequencing of the psaA gene from S. pneumoniae, serotype 6b.

A chromosomal library from S. pneumoniae serotype 6B was prepared aspreviously described (Sampson et al. 1994. Cloning and nucleotidesequence analysis of psaA, the Streptococcus pneumoniae gene encoding a37-kilodalton protein homologous to previously reported Streptococcussp. adhesins. Infect. Immun. 62:319-324.), except that pUC18 was used asthe cloning vector instead of pUC 13. Recombinants were screened bycolony immunoblot using monoclonal antibody (MAb) 1E7. (Russell et al.1990. Monoclonal antibody recognizing a species-specific protein fromStreptococcus pneumoniae J. Clin. Microbiol. 28:2191-2195). Thisprocedure, as well as plasmid purification from positive clones(Ish-Horowicz et al. 1981. Rapid and efficient cosmid cloning. NucleicAcids Res. 9:2989-2998.) and restriction endonuclease analysis, has allbeen previously described. (Sampson et al. 1990. Nucleotide sequence ofhtpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa)common antigen, formerly designated the 60-kDa common antigen. Infect.Immun. 58:3154-3157 and Sampson et al. 1994). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotanalysis were done as before (Sampson et al. 1990). All other DNAmanipulations were done according to methods described in Sambrook etal. DNA sequencing was performed using the ABI PRISM Dye TerminatorCycle Sequencing kit and procedure (Perkin-Elmer, Cetus, Foster City,Calif.). Sequence data were analyzed with the DNASTAR software program(DNASTAR Inc., Madison, Wis.) and the Wisconsin Genetics Computer Groupsequence analysis software program (Fenno et al. 1989. Nucleotidesequence analysis of a type 1 fimbrial gene of Streptococcus sanguisFW213. Infect. Immun. 57:3527-3533).

Preparation of genomic DNA for PCR-RFLP analysis. High molecular weightpneumococcal DNA was prepared by the procedure of Graves and Swaminathan(Graves et al. 1993. Universal bacterial DNA isolation procedure, p.617-621. In D. H. Pershing et al. (ed), Diagnostic molecular biology.American Society for Microbology, Washington, D.C.) with modifications.Sixteen-hour cultures of type specific S. pneumoniae were grown in 50 mlof Todd-Hewitt broth containing 0.5% yeast extract in screw cap flasksat 37° C. without shaking. Cultures were pelleted at 8000×g for 15 minat room temperature and washed with phosphate-buffered saline (10 mM pH7.2). The cell pellet was solubilized in 2.5 ml of buffer composed of 10mM Tris, 1.0 mM EDTA, pH 8.0, and 0.4% SDS. Fifteen microliters ofproteinase K (20 mg/ml) was added, and the lysate was incubated at 37°C. for 1 h. The mixture was adjusted to 0.48M NaCl with the addition of500 μl of 5M NaCl and, after mixing by inversion, 400 μl of 10%hexadecyltrimethylammonium bromide in 0.7% NaCl was added. Thissuspension was mixed as before, incubated for 30 min at 65° C., andextracted with an equal volume of phenol-chloroform-isoamyl alcohol. Theupper aqueous phase was separated by centrifugation at 1500×g andextracted with chloroform-isoamyl alcohol. DNA was precipitated from theupper aqueous phase with 2.5 volumes of ethanol at -70° C. for 30 min.It was pelleted and dried in a desiccator, resuspended in water andquantitated by measuring absorbance at 260 nm.

PCR-RFLP. Restriction enzymes EcoRI, HinfI, MaeIII, MboII, MnlI, andNheI were obtained from Boerhringer Mannheim Biochemicals (Indianapolis,Id.); RsaI, Tsp509I, Eco57I, and XmnI were purchased from New EnglandBiolabs (Beverly, Mass.). Primer sequences for the amplificationreaction were selected from the N-terminal (nucleotides 181-201) andC-terminal (nucleotides 1106-1126) sequences of the S. pneumoniaeserotype 6B gene (P1, AGGATCTAATGAAAAAATTAG (SEQ ID NO:3); P2,TCAGAGGCTTATTTTGCCAAT (SEQ ID NO:4)) and flanking regions. The primerswere synthesized at the Centers for Disease Control and Prevention usingstandard procedures.

(i) DNA amplification. The reaction was performed with the Perkin-ElmerPCR amplification kit. Reaction volumes were 100 μl and contained thestandard 1× reaction buffer without Mg, 1 μM of each primer, 2.0 mMMgCl₂, 0.2 mM dNTPs, template DNA, and 2.5 U of Taq DNA polymerase. Thesource of the template DNA was either extracted purified chromosomal DNAor a bacterial colony. Conditions for amplification were as follows:30cycles of denaturation 94° C., 1 min., annealing 52° C., 0.5 min., andextension 72° C., 1.5 min. Amplified products were separated on a 1%agarose gel and visualized with ethidium bromide. A direct colonyamplification procedure was adapted, which shortened templatepreparation by eliminating the necessity of extracting chromosomal DNA.The procedure consisted of adding a single bacterial colony directlyfrom the plate into the PCR reaction mixture and heating at 95° C. for10 minutes. The remaining PCR steps were performed as outlined forextracted chromosomal DNA and are given above.

(ii) Enzyme digestion. Digestion of amplified products was performed asdirected by the manufacturer for the designated enzymes in volumes of 20μl. Digestion products were analyzed by agarose (2% Metaphor agarose,FMC Corp., Rockland, Me.) gel electrophoresis and visualized after beingstained with ethidium bromide.

Analysis of type 6B psaA.

Genomic DNA was partially digested by Sau3AI was ligated toBamHi-digested pUC 18 and used to transform E. coli DH5α. Recombinantcolonies were selected for resistance to ampicillin and the formation ofwhite colonies in the presence of isopropyl-β-D-galactopyranoside (IPTG)and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Colony immunoblotscreening (using anti-PsaA MAb) of approximately 2,500 colonies yieldedtwo positive clones, which were selected, purified, and rescreened byWestern blot analysis using the same MAb. They both expressed a proteinreactive with MAb to PsaA and which migrated in SDS-PAGE with theexpected molecular mass of approximately 37 kDa. One was selected forcontinued study and was designated pSTR6.

Limited restriction enzyme analysis of DNA from the recombinant plasmidshowed that the positive clone contained an insert that was 3.5 kb withsites for enzymes ClaI, EcoRl, and HindIII. To localize the PsaA codingregion, the insert was double digested with SstI (multiple cloning sitein vector) and HindIII. The resultant fragments were ligated into pUC18and transformed into E. coli DH5α. This generated a recombinantcontaining an insert of ˜1.3 kb in size. The resultant subclone pSTR6y,when analyzed by SDS-PAGE and Western blot using anti-PsaA MAb, wasshown to express full length PsaA immuno-reactive protein.

The complete nucleotide sequence on both strands of the 1.3-kb insertwas determined by cycle sequencing of the plasmid subclone usingoligonucleotide primers complementary to the sequence. These were madeas sequence information became available. The nucleotide sequence of theentire streptococcal insert is set forth in the Sequence Listing as SEQID NO:1. The single open reading frame (ORF) present, beginning atnucleotide (nt) 189 and ending at nt 1117, encodes the psaA genesequence. This ORF is 930 nt long and when amplified and subcloned intovector systems such as pGEM (Promega, Madison, Wis.) and BAC-to-BAC™expression system (Bethesda Research Laboratories, Gaithersburg, Md.)expresses full-length PsaA, reactive with anti-PsaA MAb antibodies. ThisORF encodes a peptide of 309 amino acids with a deduced molecular weightof 34,598 and an isoelectric point of 5.23. Analysis of the peptideusing the algorithm of Kyte and Doolittle (Kyte et al. 1982. "A simplemethod for displaying the hydropathic character of a protein." J. Mol.Biol. 157:105-132) shows that the peptide contains a major hydrophobicregion of 20 amino acids which encodes a putative leader sequence. Thisleader contains the consensus sequence for signal peptidase cleavage(LXXC). Removal of this leader would result in a peptide of molecularmass 32,465 with a predicted isoelectric point of 4.97. A consensussequence for a ribosomal binding site (Shine et al. 1974. "The3'-terminal sequence of E. coil 16S ribosomal RNA: complementarity tononsense triplets and ribosomal binding sites." Proc. Natl. Acad. Sci.U.S.A. 71:1324-1346) is located 5 nt upstream of the ATG start codon.

Comparison of the serotype 6B sequence with streptococcal homologs

Comparison of the serotype 6B psaA nucleotide sequence ((Bilofsky et al.1988. A GenBank genetic sequence database. Nucleic Acids Res.16:1861-1864) GenBank accession number U53509) and its flanking regionswith the previously published strain R36A psaA sequence (Sampson et al.1994. "Cloning and nucleotide sequence analysis of psaA, theStreptococcus pneumoniae gene encoding a 37-kilodalton proteinhomologous to previously reported Streptococcus sp. adhesins." Infect.Immun. 62:319-324) shows the differences between the nucleotidesequences. The computed homology between the two sequences is 74%. Majorareas of discord are in regions upstream and downstream of the ORF andin the initial 60 nt which encode the putative signal peptide. When thetwo PsaA coding sequences are compared the sequence homology increasesto 78%. Serotype 6B sequence was also compared to the psaA DNA sequencefor another vaccine serotype, serotype 2, which was recently submittedto GenBank by Berry and Paton (Accession number U40786). Computeranalysis of these two sequences shows that they are very similar, withcomputed DNA homology percentages of 99% between the two psaA DNAsequences. There are eight single base differences between the twosequences.

A comparison of serotype 2 and 6B PsaAs shows almost complete identity:the computed similarity value is 99.3. The eight base difference at thenucleotide level translated into a difference at the peptide level ofsix amino acids with two of the changes resulting in conservativesubstitutions. Further analyses and comparisons of the serotype 6Bsequence to the other five GenBank PsaA homologues from viridansStreptococci and E. faecalis (Fenno et al. 1989. "Nucleotide sequenceanalysis of a type 1 fimbrial gene of Streptococcus sanguis FW213."Infect. Immun. 57:3527-3533, Sampson et al. 1994. "Cloning andnucleotide sequence analysis of psaA, the Streptococcus pneumoniae geneencoding a 37-kilodalton protein homologous to previously reportedStreptococcus sp. adhesins." Infect. Immun. 62:319-324, Ganeshkumar etal. 1991. "Nucleotide sequence of a gene coding for a salvia-bindingprotein (SsaB) from Streptococcus sanguis 12 and possible role of theprotein in coaggregation with actinomyces." Infect. Immun. 59:1093-1099,Kolenbrander et al. 1994. "Nucleotide sequence of the Streptococcusgordonii PK488 coaggregation adhesin gene scaA and ATP-bindingcassette." Infect. Immun. 62:4469-4480, and Lowe et al. 1995. "Cloningof an Enterococcus faecalis endocarditis antigen: homology with someadhesins from oral streptococci. " Infect. Immun 63:703-706) revealedsignificant sequence similarity between them. Sequence identities were81%, 81%, 77%, 82%, and 57%, respectively, for PsaA (S. pneumoniaestrain R36A), SsaB (S. sanguis), FimA (S. parasanguis), ScaA (S.gordonii) and EfaA (E. faecalis). Additionally, all six sequences showedgreat similarity in organization. They have a hydrophobic leader peptidecontaining the prolipoprotein consensus sequence LXXC (for signalpeptidase II cleavage) within the first 17-20 amino acids. ThisN-terminal leader sequence appears to represent the area of greatestvariability. It is followed by a region of high similarity from aminoacids 36-150. The region from 150 to 198 is a variable region and isfollowed by another conserved region (198-309).

PCR-RFLP analysis of chromosomal DNA from the 23 serotype strains in a23-valent vaccine.

PCR-RFLP was used to examine the degree of conservation of the geneamong 23 S. pneumoniae serotypes, representing the 23 serotypes in a23-valent vaccine. Since previous attempts to amplify pneumococcal typestrains with primers corresponding to strain R36A were unsuccessful,primers for PCR were selected from N-terminal and C-terminal sequencesof serotype 6B. Using primers complementary to serotype 6B, the psaAgene from all 23 serotypes and subtypes represented in the 23-valentvaccine was amplified from chromosomal DNA. A total of 10 enzymes werechosen that had restriction endonuclease digestion sites throughout theentire length of the serotype 6B psaA gene. Nine of the 10 enzymes gaveidentical patterns for all 23 psaA genes analyzed.

Cleavage with restriction enzyme Tsp509I was the one exception to thoseenzymes that generated identical patterns. Tsp509I has six sites withinthe gene and generates seven fragments upon digestion with sizes of 7,30, 68, 146, 151, 166, and 362 bp. When these fragments are separated on2% metaphor agarose gel, a five-band pattern can be seen (7- and 30-bpfragments are not seen on these gels because of their small size). For21 of 23 serotypes this five-fragment enzyme pattern was obtained; butfor strains of serotype 4 and 33F, the 146-bp fragment is absent and twonew fragments appear flanking the 68-bp fragment making a total of sevenbands. This increase in fragment number results from the presence of anextra Tsp509I site within the 146-bp fragment.

To ascertain the prevalence of this extra site, the Tsp509I patterns of3 to 4 additional strains of each of 23 serotype strains (additionalstrains of serotype 2 and serotype 25 were not available) were analyzed.All strains analyzed were random clinical isolates from the UnitedStates that had been submitted to CDC for serotyping. The majority ofthe 80 strains were blood isolates; exceptions were 2 from cerebrospinalfluid, 2 from pleural fluid, and 1 each from the eye and nose. Of thestrains analyzed, 10% had the extra Tsp509I site, resulting in thealtered RFLP pattern. This modification was seen only in types 4, 8,11F, and 33F. In an attempt to determine prevalence of this alteredpattern, we analyzed the psaA gene from 8 additional strains of these 4types for the Tsp509I variation (bringing the total to 11-12 for these 4types). Table 1 summarizes the analyses of serotypes 4, 8, 11A, and 33F;it shows that the modified pattern is randomly present in 4 and 8, butis present in 11 of 12 strains of 11A and all strains of 33F. Theoccurrence of this pattern could not be correlated with geographiclocation or region of the United States since strains that showedvariation came from diverse regions of the country. All strains of types4, 8, 11A, and 33F were blood isolates except one 33F strain, which wasa nasal isolate; thus the relevance of the site of isolation onprevalence of this modification could not be assessed.

                  TABLE 1                                                         ______________________________________                                        Screening of selected serotypes for additional Tsp509I restriction site       Ratio of serotypes with additional                                                                   Total serotypes with                                   site to total no. of serotypes tested                                                                unique patterns                                        Serotype                                                                             Expt. #1.sup.a                                                                            Expt. #2.sup.b                                                                            % Unique pattern                               ______________________________________                                         4     1/3         3/9         .sup. 33 (4/12).sup.c                           8     3/4         4/9         44 (7/13)                                      11A    2/3         9/9          92 (11/12)                                    33F    3/3         9/9         100 (12/12)                                    ______________________________________                                         .sup.a Initial Tsp509I analysis which included survey of 2-3 strains each     of all 23 vaccine types.                                                      .sup.b Tsp509I analysis of more strains of types showing additional           Tsp509I site.                                                                 .sup.c Shown in parenthesis is ratio of number with additional site to        number tested.                                                           

This analysis discloses the cloning and sequencing of the gene encodingPsaA from S. pneumoniae serotype 6B and a subsequent analysis of thegene in the 23 pneumococcal polysaccharide vaccine serotypes. Sequenceanalysis revealed that the serotype 6B sequence and the previouslypublished strain R36A were less similar than expected. The nucleotidesequence and its flanking regions were only 73% homologous to theoriginal strain R36A psaA, with the actual PsaA coding sequences had acomputed homology of 78%. Protein sequence similarity between the twosequences was only 81%. A comparison of the serotype 6B sequence withthe newly submitted serotype 2 pneumococcal psaA (a vaccine serotype)gave computed DNA homology values of 99% and 98% protein sequencesimilarity. These values are evidence of the high sequence conservationfor the gene within the vaccine serotypes. Moreover, when the deducedamino acid sequences of these two sequences were compared with otherpublished sequences for PsaA homologues within the genus, large areas ofsimilarity were evident for all five proteins. Similarity values withinthe group ranged from 57% to 82%.

The need for a Streptococcus pneumoniae vaccine candidate prompted us toclone and sequence the psaA gene from S. pneumoniae serotype 6B. Theheterogeneity between the two pneumococcal psaA genes (6B and R36A) ledus to examine the vaccine serotypes to determine the degree of diversityamong strains. Primers homologous with the N terminus and C terminus ofthe serotype 6B sequence amplified all 23 of the vaccine serotypes.PCR-RFLP analysis using 10 different restriction enzymes representing 21sites within the serotype 6B gene and shows only one area of diversity,which resulted in an additional Tsp509I site for a small number ofstrains.

This study demonstrates that the serotype 6B gene sequence isrepresentative of the sequence found among the vaccine serotypes.Evidence for this includes the 99% DNA sequence identity betweenserotype 2 and serotype 6B and the uniform and identical restrictionpatterns covering the 21 sites examined in this study. It is clear thatour earlier strain R36A psaA sequence represents a variant sequenceseemingly not present in the serotypes that were analyzed here since wewere unable to amplify them using primers to strain R36A psaA.

The more important aspect of this study, however, is that there islimited diversity among the vaccine serotypes analyzed. These are theserotypes that cause disease and thus, the ones against whichprophylactic measures are needed. The lack of genetic diversity of psaAamong these serotypes suggests that gene is highly conserved and is anexcellent candidate for vaccine development.

The 37-kDa protein from serotype 22F was used to generate monoclonalantibodies 1B6E12H9, 3C4D5C7, 4E9G9D3, 4H5C10F3, 6F6F9C8, 8G12G11B10which were analyzed for their ability to confer protection frominfection by Streptococcus pneumoniae. Table 2 shows that of 5monoclonal antibodies tested, one in particular gave efficientprotection from subsequent S. pneumoniae challenge (8G12G11B10). Theprotection from S. pneumoniae was dose-responsive, demonstrating thatthe monoclonal antibody was responsible for the protection (Table3).

                  TABLE 2                                                         ______________________________________                                        Passive protection of five (5) Anti-37 kDa murine monoclonal                  antibodies in an infant mouse model to                                        Streptococcus pneumoniae serotype 6B.                                                            Death                                                      37 kDa MAb Bacteremia    @ 48 h  @ 14 d                                       Cell Line.sup.a                                                                          @ 48 h (%)    (%)     (%)                                          ______________________________________                                        1E7. . .   100           100     100                                          8G12. . .  100            0       20                                          4E9. . .   100           80      100                                          6F6. . .   100           60      100                                          1B6. . .   100           80      100                                          ______________________________________                                         .sup.a Challenge does (1.7 × 10.sup.3 cfu) or 10X bacteremic dose       100% (BD.sub.100).                                                            Five/mice group given 50 μg total antibody. All MAbs are IgG.         

                  TABLE 3                                                         ______________________________________                                        Effect of a Second Dose on the Passive Protective Potential                   of the Anti-37 kDa Murine Monoclonal Antibody 8G12.                           Ab Dose Level                                                                             Bacteremia         Death                                          (μg)     @ 48h              @ 48 h.                                                                             @ 10 d                                   Pre    Post.sup.a                                                                             %      Ave cfu/ml                                                                              (%)   (%)                                    ______________________________________                                        50     --       100    1.2 × 10.sup.4                                                                     0    30                                     50     50        80    1.0 × 10.sup.4                                                                     0    50                                      5     --       100    4.7 × 10.sup.4                                                                    70    100                                     5     5        100    3.0 × 10.sup.4                                                                    50    80                                     --     --       100    >10.sup.5 80    100                                    ______________________________________                                         .sup.a All infant mice were challenged with 10X BC.sub.100 (2 ×         10.sup.3 cfu). Ab given 24 h prior to and 24 h after (post) challenge. 10     mice/group.                                                              

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1330 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 189..1115                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TACTGCTTCAGTTTTGGGACTCTTTATTGGCTATAGTTTTAATGTTGCGGCAGGTTCTAG60                TATCGTGCTTACAGCTGCTAGTTTCTTTCTCATTAGCTTCTTTATCGCTCCCAAACAACG120               ATATTTGAAACTGAAAAATAAACATTTGTTAAAATAAGGGGCAAAGCCCTAATAAATTGG180               AGGATCTAATGAAAAAATTAGGTACATTACTCGTTCTCTTTCTTTCTGCA230                         MetLysLysLeuGlyThrLeuLeuValLeuPheLeuSerAla                                    1510                                                                          ATCATTCTTGTAGCATGTGCTAGCGGAAAAAAAGATACAACTTCTGGT278                           IleIleLeuValAlaCysAlaSerGlyLysLysAspThrThrSerGly                              15202530                                                                      CAAAAACTAAAAGTTGTTGCTACAAACTCAATCATCGCTGATATTACT326                           GlnLysLeuLysValValAlaThrAsnSerIleIleAlaAspIleThr                              354045                                                                        AAAAATATTGCTGGTGACAAAATTGACCTTCATAGTATCGTTCCGATT374                           LysAsnIleAlaGlyAspLysIleAspLeuHisSerIleValProIle                              505560                                                                        GGGCAAGACCCACACGAATACGAACCACTTCCTGAAGACGTTAAGAAA422                           GlyGlnAspProHisGluTyrGluProLeuProGluAspValLysLys                              657075                                                                        ACTTCTGAGGCTGATTTGATTTTCTATAACGGTATCAACCTTGAAACA470                           ThrSerGluAlaAspLeuIlePheTyrAsnGlyIleAsnLeuGluThr                              808590                                                                        GGTGGCAATGCTTGGTTTACAAAATTGGTAGAAAATGCCAAGAAAACT518                           GlyGlyAsnAlaTrpPheThrLysLeuValGluAsnAlaLysLysThr                              95100105110                                                                   GAAAACAAAGACTACTTCGCAGTCAGCGACGGCGTTGATGTTATCTAC566                           GluAsnLysAspTyrPheAlaValSerAspGlyValAspValIleTyr                              115120125                                                                     CTTGAAGGTCAAAATGAAAAAGGAAAAGAAGACCCACACGCTTGGCTT614                           LeuGluGlyGlnAsnGluLysGlyLysGluAspProHisAlaTrpLeu                              130135140                                                                     AACCTTGAAAACGGTATTATTTTTGCTAAAAATATCGCCAAACAATTG662                           AsnLeuGluAsnGlyIleIlePheAlaLysAsnIleAlaLysGlnLeu                              145150155                                                                     AGCGCCAAAGACCCTAACAATAAAGAATTCTATGAAAAAAATCTCAAA710                           SerAlaLysAspProAsnAsnLysGluPheTyrGluLysAsnLeuLys                              160165170                                                                     GAATATACTGATAAGTTAGACAAACTTGATAAAGAAAGTAAGGATAAA758                           GluTyrThrAspLysLeuAspLysLeuAspLysGluSerLysAspLys                              175180185190                                                                  TTTAATAAGATCCCTGCTGAAAAGAAACTCATTGTAACCAGCGAAGGA806                           PheAsnLysIleProAlaGluLysLysLeuIleValThrSerGluGly                              195200205                                                                     GCATTCAAATACTTCTCTAAAGCCTATGGTGTCCCAAGTGCCTACATC854                           AlaPheLysTyrPheSerLysAlaTyrGlyValProSerAlaTyrIle                              210215220                                                                     TGGGAAATCAATACTGAAGAAGAAGGAACTCCTGAACAAATCAAGACC902                           TrpGluIleAsnThrGluGluGluGlyThrProGluGlnIleLysThr                              225230235                                                                     TTGGTTGAAAAACTTCGCCAAACAAAAGTTCCATCACTCTTTGTAGAA950                           LeuValGluLysLeuArgGlnThrLysValProSerLeuPheValGlu                              240245250                                                                     TCAAGTGTGGATGACCGTCCAATGAAAACTGTTTCTCAAGACACAAAC998                           SerSerValAspAspArgProMetLysThrValSerGlnAspThrAsn                              255260265270                                                                  ATCCCAATCTACGCACAAATCTTTACTGACTCTATCGCAGAACAAGGT1046                          IleProIleTyrAlaGlnIlePheThrAspSerIleAlaGluGlnGly                              275280285                                                                     AAAGAAGGCGACAGCTACTACAGCATGATGAAATACAACCTTGACAAG1094                          LysGluGlyAspSerTyrTyrSerMetMetLysTyrAsnLeuAspLys                              290295300                                                                     ATTGCTGAAGGATTGGCAAAATAAGCCTCTGAAAAACGTCATTCTCATGTG1145                       IleAlaGluGlyLeuAlaLys                                                         305                                                                           AGCTGGCGTTTTTTCTATGCCCACATTTCCGGTCAAATCATTGGAAAATTCTGACTGTTT1205              CAGATACAATGGAAGAAAAAAGATTGGAGTATCCTATGGTAACTTTTCTCGGAAATCCTG1265              TGAGCTTTACAGGTAAACAACTACAAGTCGGCGACAAGGCGCTTGATTTTTCTCTTACTA1325              CAACA1330                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysLysLeuGlyThrLeuLeuValLeuPheLeuSerAlaIleIle                              151015                                                                        LeuValAlaCysAlaSerGlyLysLysAspThrThrSerGlyGlnLys                              202530                                                                        LeuLysValValAlaThrAsnSerIleIleAlaAspIleThrLysAsn                              354045                                                                        IleAlaGlyAspLysIleAspLeuHisSerIleValProIleGlyGln                              505560                                                                        AspProHisGluTyrGluProLeuProGluAspValLysLysThrSer                              65707580                                                                      GluAlaAspLeuIlePheTyrAsnGlyIleAsnLeuGluThrGlyGly                              859095                                                                        AsnAlaTrpPheThrLysLeuValGluAsnAlaLysLysThrGluAsn                              100105110                                                                     LysAspTyrPheAlaValSerAspGlyValAspValIleTyrLeuGlu                              115120125                                                                     GlyGlnAsnGluLysGlyLysGluAspProHisAlaTrpLeuAsnLeu                              130135140                                                                     GluAsnGlyIleIlePheAlaLysAsnIleAlaLysGlnLeuSerAla                              145150155160                                                                  LysAspProAsnAsnLysGluPheTyrGluLysAsnLeuLysGluTyr                              165170175                                                                     ThrAspLysLeuAspLysLeuAspLysGluSerLysAspLysPheAsn                              180185190                                                                     LysIleProAlaGluLysLysLeuIleValThrSerGluGlyAlaPhe                              195200205                                                                     LysTyrPheSerLysAlaTyrGlyValProSerAlaTyrIleTrpGlu                              210215220                                                                     IleAsnThrGluGluGluGlyThrProGluGlnIleLysThrLeuVal                              225230235240                                                                  GluLysLeuArgGlnThrLysValProSerLeuPheValGluSerSer                              245250255                                                                     ValAspAspArgProMetLysThrValSerGlnAspThrAsnIlePro                              260265270                                                                     IleTyrAlaGlnIlePheThrAspSerIleAlaGluGlnGlyLysGlu                              275280285                                                                     GlyAspSerTyrTyrSerMetMetLysTyrAsnLeuAspLysIleAla                              290295300                                                                     GluGlyLeuAlaLys                                                               305                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGGATCTAATGAAAAAATTAG21                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TCAGAGGCTTATTTTGCCAAT21                                                       __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid whose nucleotidesequence encodes a protein of Streptococcus pneumoniae, the proteinbeing the 37-kDa surface adhesin A protein whose amino acid sequence isset forth in SEQ ID NO:2.
 2. The isolated nucleic acid of claim 1,wherein the nucleic acid is the nucleic acid set forth in SEQ ID NO:1.3. An isolated nucleic acid comprising at least 50 contiguousnecleotides of the nucleic acid set forth in SEQ ID NO:1.
 4. An isolatednucleic acid comprising the nucleic acid set forth in SEQ ID NO:3.
 5. Anisolated nucleic acid comprising the nucleic acid set forth in SEQ IDNO:4.
 6. The isolated nucleic acid of claim 1 in a vector appropriatefor expressing the nucleic acid.
 7. The vector of claim 6 in a host cellappropriate for expressing the nucleic acid.
 8. The isolated nucleicacid of claim 3 in a vector appropriate for expressing the nucleic acid.9. The vector of claim 8 in a host cell appropriate for expressing thenucleic acid.